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Determination of the Orientation and Dynamics of Ergosterol in Model Membranes Using Uniform ¹³C Labeling and Dynamically Averaged ¹³C Chemical Shift Anisotropies as Experimental Restraints

O. Soubias ^*, F. Jolibois , S. Massou , A. Milon ^* and V. Réat ^*

^* Institut de Pharmacologie et de Biologie Structurale, UMR 5089, Toulouse, France; Laboratoire de Physique Quantique, UMR 5626, Institut de Recherché sur les Systèmes Atomiques et Moléculaires Complexes, Université P. Sabatier, Toulouse, France; and Service de Résonance Magnétique Nucléaire, Fédération de Recherche Toulousaine en Chimie Moléculaire, Université P. Sabatier, Toulouse, France

Correspondence: Address reprint requests to Valerie Réat, Tel.: 33-5-6117-5418; Fax: 33-5-6117-5424; E-mail: valerie.reat{at}ipbs.fr.

A new strategy was established to determine the average orientation and dynamics of ergosterol in dimyristoylphosphatidylcholine model membranes. It is based on the analysis of chemical shift anisotropies (CSAs) averaged by the molecular dynamics. Static ¹³C CSA tensors were computed by quantum chemistry, using the gauge-including atomic-orbital approach within Hartree-Fock theory. Uniformly ¹³C-labeled ergosterol was purified from Pichia pastoris cells grown on labeled methanol. After reconstitution into dimyristoylphosphatidylcholine lipids, the complete ¹H and ¹³C assignment of ergosterol's resonances was performed using a combination of magic-angle spinning two-dimensional experiments. Dynamically averaged CSAs were determined by standard side-band intensity analysis for isolated ¹³C resonances (C₃ and ethylenic carbons) and by off-magic-angle spinning experiments for other carbons. A set of 18 constraints was thus obtained, from which the sterol's molecular order parameter and average orientation could be precisely defined. The validity of using computed CSAs in this strategy was verified on cholesterol model systems. This new method allowed us to quantify ergosterol's dynamics at three molar ratios: 16 mol % (Ld phase), 30 mol % (Lo phase), and 23 mol % (mixed phases). Contrary to cholesterol, ergosterol's molecular diffusion axis makes an important angle (14°) with the inertial axis of the rigid four-ring system.